Transcription factors RUNX1 and CBFbeta play key roles in leukemogenesis and normal hematopoiesis. Mutations or chromosome translocations affecting RUNX1 or CBFB (which encodes CBFbeta) are found in 20-30% of patients with acute leukemia. A chromosome 16 inversion inv(16) that generates a fusion gene between CBFB and MYH11 (which encodes the smooth muscle myosin heavy chain, SMMHC) is found in all patients with acute myeloid leukemia (AML) subtype M4Eo. My lab has studied RUNX1 and CBFbeta for their roles in leukemogenesis and normal hematopoiesis for the last 16 years. We have established a mouse model of human AML by targeted insertion of the fusion gene CBFB-MYH11 in mouse ES cells, which demonstrated the critical role of CBFB-MYH11 in leukemogenesis. Using transgenic mouse and zebrafish models we have demonstrated that RUNX1 and CBFbeta are required for multiple steps of normal hematopoiesis, starting from the hematopoietic stem cells. We have also conducted in vitro analysis to understand the molecular level mechanisms of CBFbeta-SMMHC function, which will be critical for designing new therapeutic approaches for AML. Our section has been using transgenic mouse models to study the mechanism of leukemogenesis by the fusion gene CBFB-MYH11 and its related RUNX1 gene. In the past year we were able to generate data demonstrating cooperation between CBFB-MYH11 and mutated KIT gene for leukemogenesis. KIT mutations is relatively common in human patients with leukemia, and our data suggest that KIT mutations contribute to leukemia development and are therefore potential targets for therapy. We also provided evidence that the interaction between CBFB-MYH11 and RUNX1 is critical for the leukemogenic function of CBFB-MYH11. Likewise, RUNX1-CBFbeta interaction might be critical for leukemia involving RUNX1 mutations, such as the AML1-ETO (also known as RUNX1-ETO) fusion gene generated by t(8;21) in AML. Thus, inhibitors of CBFbeta - RUNX1 interaction may have potential therapeutic applications for both (inv)16 and t(8;21) AML, which account for 20-30% of all AML cases. In collaboration with the NIH Chemical Genomics Center (NCGC), we developed a CBFbeta and RUNX1 bead-based proximity assay in Amplified Luminescence Proximity Homogenous Assay (ALPHA) Screen format and optimized it for high throughput screening. A total of 243,398 compounds were screened with this assay at NCGC, which led to the identification of 137 putative inhibitors by Structure-Activity Relationships and Curve Class. Confirmatory ALPHA and HTRF (homogeneous time resolved fluorescence) assays were performed and candidate compounds showing consistent results were further tested by Biacore to characterize the kinetics and binding affinity of the compounds. These follow-up tests have so far identified 70 potential candidate compounds. Three related lead hits have been confirmed in tissue culture cells and in our zebrafish model. Importantly, at least one of the three compounds reduced leukemia burden in a mouse CBF leukemia model, with comparable efficacy to and synergistic with cytarabine. These findings may lead to the development of targeted therapy for CBF leukemias.